Enzyme immunoassay (EIA) is an assay technique based on two facts: (1) the immune system of vertebrates can produce an almost unlimited variety of antibodies each with a specific affinity for an antigen, and (2) the high catalytic power and specificity of enzymes, which it often is quite easy to detect. In the EIA technique, the immuno-reactants, the antibody and the corresponding antigen are reacted with each other and this reaction is detected using enzymes labelled to one of the immuno-reactants. Different enzymes are used in the assay and among these are .beta.-galactosidase, peroxidase, e.g. horseradish peroxidase, and alkaline phosphatase. For the detection of the enzyme activity is used a chromogenic substrate and a chromogen which develop a colour during the reaction. This technique is very useful both for diagnostic purposes and in basic research work. The enzyme immunoassays have several advantages. It is possible to obtain very high sensitivity and specificity with relatively cheap reagents and equipment. The methods to produce monoclonal antibodies have enhanced the possibility of standardisation of EIA with even higher sensitivity and specificity and contributes to new assay designs.
A modification of the EIA and one of the very widely used enzyme immunoassay techniques today is the ELISA (enzyme-linked-immunosorbent-assay) which can be used for both qualitative and quantitative assays. In principle an enzyme is coupled to an antibody against the antigen to be determined. The assay is usually performed in trays of polystyrene or polypropylene to which the antigen is immobilised. The enzyme is chemically coupled to the antibody and incubated with a substrate solution containing a suitable buffer, a chromogenic substrate, e.g. hydrogen peroxide or urea peroxide, and a chromogen which changes colour when the peroxide is oxidised. There are many variations of an ELISA. In competitive assays, the enzyme may be coupled with the antigen. The number of "layers" of antigen and antibodies may vary as well as it is possible to use different enzymes. In ELISA techniques, horseradish peroxidase (HRP) is a commonly used enzyme. For the detection of the enzyme activity is needed a specific enzyme substrate for the actual enzyme. Various enzyme substrates such as ortho-phenylenediamine (OPD), 2,2'-azino-bis-(3-ethylbenz-thiazoline-6-sulfonic acid) (ABTS) and 3,3',5,5'-tetramethylbenzidine (TMB) are commonly used. TMB is a non-hazardous normally colourless substrate, which forms an intensely blue coloured oxidation product in the presence of peroxide and HRP. TMB is often preferred as substrate because it is at this time the most sensitive substrate for HRP and is less toxic than e.g. OPD.
When performing an ELISA assay it is important to reduce the background signal in order to obtain the best possible sensitivity. It has therefore been considered important that the substrate components themselves should be colourless or only very slightly coloured in order for them to be used as substrate in an ELISA assay. Such properties will minimise the blind absorbance values. This means that pipetting of the thus colourless substrate into the enzyme immunoassay reaction vessel, which often is a transparent tube or a transparent ELISA tray, is associated with the risk of incorrect dosage in that it may be difficult to determine immediately by visual examination whether the substrate has been added to a vessel (e.g. a well) or not. As a consequence, the substrate may not be added to all vessels, or twice the required amount of substrate may be added to some vessels. In automated assays it is not easily achievable to make control measurements to ensure that the substrate or the proper amount of substrate has been added.
German Patent No. DE 195 27 160 C1 describes a method for photometrically measuring of the concentration of an enzyme by measuring a colour created by enzymatic reaction with a colourless substrate. The enzymatic reaction is stopped by adding 2 N NaOH. To control the addition of stopping solution a dye (Amaranth) is included in the otherwise colourless solution. The German patent relates to the inclusion of a dye in a stop solution, but does not address the problems of storage stability and construction of water-based substrate systems if a dye should be included in such systems for the same purpose.
U.S. Pat. No. 4,128,629 describes a method for determining small concentrations of cortisol by using an immunoassay technique, more specific a radio-immuno-assay (RIA). In an example the possibility of including a non-interfering red dye to indicate the presence of the tracer used in the assay is mentioned. There is no mentioning of enzyme immunoassays and the specific problems associated to the stabilisation and storage stability of substrate solutions having dyes included therein.
U.S. Pat. No. 5,318,894 describes a dry phase test device and method for determining the presence of a peroxidatively active substance. The test device includes a test pad comprising a suitable carrier matrix with an indicator reagent. The indicator reagent composition for impregnation of the test area of the test device includes an indicator dye (e.g. TMB), a hydroperoxide and a promoter. To improve the colour resolution and differentiation one or more inert background dyes can be included in the indicator reagent. The amount of organic solvent present in the indicator reagent for impregnation is up to 90%. U.S. Pat. No. 5,318,894 does however not address the problems with respect to preparation and storage stability of a water-based TMB substrate solution system. Storage stability of the liquid compositions does not appear to be relevant in that the liquid compositions are used immediately after preparation. Also, the dyes used in U.S. Pat. No. 5,318,894 appears to be selected so that they deliberately have a high absorbance in the same range as the reacted TMB substrate.